The present invention relates to a compound.
In particular the present invention relates to a compound and to a pharmaceutical composition comprising the compound. The present invention also relates to the use of that compound in the field of medicine.
Evidence suggests that oestrogens are the major mitogens involved in promoting the growth of tumours in endocrine-dependent tissues, such as the breast and endometrium. Although plasma oestrogen concentrations are similar in women with or without breast cancer, breast tumour oestrone and oestradiol levels are significantly higher than in normal breast tissue or blood. In situ synthesis of oestrogen is thought to make an important contribution to the high levels of oestrogens in tumours and therefore specific inhibitors of oestrogen biosynthesis are of potential value for the treatment of endocrine-dependent tumours.
Over the past two decades, there has been considerable interest in the development of inhibitors of the aromatase pathway which converts the androgen precursor androstenedione to oestrone. However, there is now evidence that the oestrone sulphatase (E1-STS) pathway, i.e. the hydrolysis of oestrone sulphate to oestrone (E1S to E1), as opposed to the aromatase pathway, is the major source of oestrogen in breast tumours. This theory is supported by a modest reduction of plasma oestrogen concentration in postmenopausal women with breast cancer treated by aromatase inhibitors, such as aminoglutethimide and 4-hydroxyandrostenedione and also by the fact that plasma E1S concentration in these aromatase inhibitor-treated patients remains relatively high. The long half-life of E1S in blood (10-12 h) compared with the unconjugated oestrogens (20 min) and high levels of steroid sulphatase activity in liver and, normal and malignant breast tissues, also lend support to this theory.
PCT/GB92/01587 teaches novel steroid sulphatase inhibitors and pharmaceutical compositions containing them for use in the treatment of oestrone dependent tumours, especially breast cancer. These steroid sulphatase inhibitors are sulphamate esters. Examples of such inhibitors are sulphamate ester derivatives of steroids.
As is well known in the art, steroids have the general formula of: 
In the above formula, the ring components have been labelled in the conventional manner.
A preferred compound of PCT/GB92/101587 is oestrone-3-sulphamate (otherwise known as xe2x80x9cEMATExe2x80x9d), which has the following structure: 
It is known that EMATE is a potent E1-STS inhibitor as it displays more than 99% inhibition of E1-STS activity in intact MCF-7 cells at 0.1 xcexcM. EMATE also inhibits the E1-STS enzyme in a time- and concentration-dependent manner, indicating that it acts as an active site-directed inactivator.
Although EMATE was originally designed for the inhibition of E1-STS, it also inhibits dehydroepiandrosterone sulphatase (DHA-STS), which is an enzyme that is believed to have a pivotal role in regulating the biosynthesis of the oestrogenic steroid androstenediol.
Also, there is now evidence to suggest that androstenediol may be of even greater importance as a promoter of breast tumour growth. EMATE is also active in vivo as almost complete inhibition of rat liver E1-STS (99%) and DHA-STS (99%) activities resulted when it is administered either orally or subcutaneously. In addition, EMATE has been shown to have a memory enhancing effect in rats. Studies in mice have suggested an association between DHA-STS activity and the regulation of part of the immune response. It is thought that this may also occur in humans. The bridging O-atom of the sulphamate moiety in EMATE is important for inhibitory activity. Thus, when the 3-O-atom is replaced by other heteroatomsxe2x80x94as in oestrone-3-N-sulphamate and oestrone-3-S-sulphamatexe2x80x94these analogues are weaker non-time-dependent inactivators.
Although optimal potency for inhibition of E1-STS may have been attained in EMATE, it is possible that oestrone may be released during sulphatase inhibition, and that EMATE and its oestradiol congener may possess oestrogenic activity.
The present invention seeks to provide novel compounds suitable for the inhibition of E1-STS but preferably wherein those compounds also have an oestrogenic effect.
Certain aspects of the present invention are presented in the accompanying claims.
A key advantage of the present invention is that the sulphamate compounds of the present invention can act as E1-STS inhibitors.
Another advantage of the compounds of the present invention is that they may be potent in vivo.
In addition, the compounds of the present invention can be used as oestrogenic compounds. Preferably some of the compounds are potent oestrogenic compounds. More preferably some of the compounds are highly oestrogenic compounds.
In a preferred embodiment therefore, the present invention therefore provides sulphamate compounds which are both steroid sulphatase inhibitors and oestrogenic.
The compounds of the present invention are also advantageous in that they may be orally active.
The sulphamate compounds of the present invention are believed to be useful for the treatment of breast cancer, or endocrine-dependent cancers, or endocrine- or oestrogen-dependent conditions and/or illnesses and/or cancers; see also documents cited herein (compounds therein also so useful).
In addition, the sulphamate compounds of the present invention are useful for the treatment of non-malignant conditions, such as the prevention of auto-immune diseases, particularly when pharmaceuticals may need to be administered from an early age.
The sulphamate compounds of the present invention are also believed to have therapeutic uses other than for the treatment of endocrine-dependent cancers, such as the treatment of autoimmune diseases and hormone replacement therapy.
The sulphamate compounds of the present invention are also believed to be useful for birth control etc.
These and further aspects of the present invention are now described.
Some or all of the ring components may be fused together or joined via one or more suitable spacer groups. The present invention also encompasses combinations thereof.
The term xe2x80x9csulphamatexe2x80x9d as used herein includes an ester of sulphamic acid, or an ester of an N-substituted derivative of sulphamic acid, or a salt thereof.
Preferably, the sulphamate group has the formula: 
wherein each of R1 and R2 is independently selected from H or a hydrocarbyl group.
The term xe2x80x9chydrocarbyl groupxe2x80x9d as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non-limiting example of a hydrocarbyl group is an acyl group.
In one preferred embodiment of the present invention, the hydrocarbyl group is a hydrocarbon group.
Here the term xe2x80x9chydrocarbonxe2x80x9d means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Preferably, R1 and R2 are independently selected from H or alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted compounds of this invention may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R1 and/or R2 is alkyl, the preferred values are those where R1 and R2 are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R1 and R2 are both methyl. When R1 and/or R2 is aryl, typical values are phenyl and tolyl (xe2x80x94PhCH3; o-, m- or p-). Where R1 and R2 represent cycloalyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R1 and R2 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94 to provide a 5-, 6- or 7-membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl and aryl we include substituted groups containing as substituents therein one or more groups which do not interfere with the sulphatase inhibitory activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl.
In some preferred embodiments, at least one of R1 and R2 is H.
The polycyclic compound can comprise at least two ring components, or least three ring components, or least four ring components.
Preferably, the polycyclic compound comprises four ring components.
Preferably the polycyclic compound will contain, inclusive of all substituents, no more than 50 about carbon atoms, more usually no more than about 30 to 40 carbon atoms.
Preferred polycyclic compounds are those that are based on steroidal ring structures, that is to say a cyclopentanophenanthrene skeleton.
Thus, a preferred polyclic compound of the present invention has a structure similar to a steroidal structure but wherein an oxime group is attached to or is part of the D ring.
In this regard, the structure of the preferred polycyclic compound can be presented as: 
wherein ring D1 represents the combination of a ring and the oxime group (i.e. the oxime group is part of or is attached to the ring component). The ring of D1 may be substituted or unsubstituted, saturated or unsaturated. The rings A, B and Cxe2x80x94which are similar to those of a steroidal nucleusxe2x80x94may be substituted or unsubstituted, saturated or unsaturated.
A preferred example of D1 has the formula: 
wherein the )xe2x80x94 symbols represent the ring of D1 being joined to the remainder of the steroidal structural formula; and X represents H or a suitable substituent. An example of a suitable substituent is a hydrocarbyl group.
A further preferred example of D1 has the formula: 
wherein the )xe2x80x94 symbols represent the ring of D1 being joined to the remainder of the steroidal structural formula; and X represents H or a suitable substituent. An example of a suitable substituent is a hydrocarbyl group. Here the indicated Me group is vertical.
As indicated, the ring of D1 may be substituted with suitable groupsxe2x80x94such as alkyl, hydroxy, halo etc. However, in a preferred embodiment, the ring atoms of D1 that are not associated with neighbouring ring(s) are unsubstituted. By way of example these ring atoms are indicated below as 1 and 2 for the above presented preferred formula: 
In compounds of the present invention the oxime group can be of either geometrical isomeric form. For example, the oxime group can be the syn isomer. In a preferred embodiment, the oxime group is the anti isomer. By way of example, the above presented formulae for D1 show the anti isomeric structure.
Thus, the present invention encompasses compounds that have geometrical isomers. The compounds may be present in just one isomeric form or in combinations thereof. In one preferred embodiment, the compound is present as at least the anti isomer. In another preferred embodiment, the compound is present only as the anti isomer.
Examples of suitable preferred steroidal nuclei rings A-C for the rings A-C of the compounds of the present invention include rings A-C of oestrone and dehydroepiandrosterone.
Preferred steroidal nuclei having suitable rings A-C for the rings A-C of the compounds of the present invention are:
oestrones and substituted oestrones, viz:
oestrones
2-OH-oestrone
2-methoxy-oestrone
4-OH-oestrone
6xcex1-OH-oestrone
7xcex1-OH-oestrone
16xcex1-OH-oestrone
16b-OH-oestrone
oestradiols and substituted oestradiols, viz:
2-OH-17xcex2-oestradiol
2-methoxy-17xcex2-oestradiol
4-OH-17xcex2-oestradiol
6xcex1-OH-17xcex2-oestradiol
7xcex1-OH-17xcex2-oestradiol
16xcex1-OH-17xcex1-oestradiol
16xcex2-OH-17xcex1-oestradiol
16xcex2-OH-17xcex2-oestradiol
17xcex1-oestradiol
17xcex2-oestradiol
17xcex1-thinyl-17xcex2-oestradiol
oestriols and substituted oestriols, viz:
oestriol
2-OH-oestriol
2-methoxy-oestriol
4-OH-oestriol
6xcex1-OH-oestriol
7xcex1-OH-oestriol
dehydroepiandrosterones and substituted dehydroepiandrosterones, viz:
dehydroepiandrosterones
6xcex1-OH-dehydroepiandrosterone
7xcex1-OH-dehydroepiandrosterone
16xcex1-OH-dehydroepiandrosterone
16xcex2-OH-dehydroepiandrosterone
In general terms the ring system ABCD1 may contain a variety of non-interfering substituents. In particular, the ring system ABCD1 may contain one or more of hydroxy, alkyl especially lower (C1-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C1-C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
In an alternative embodiment, the polyclic compound may not contain or be based on a steroid nucleus. In this regard, the polyclic compound may contain or be based on a non-steroidal ring systemxe2x80x94such as diethylstilboestrol, stilboestrol and other ring systems.
In formula (I), the at least one sulphamate group is attached to any one or more of the ring components.
Preferably, the polycyclic compound has a steroidal structure and wherein the sulphamate group is attached to the A ring.
Preferably, the sulphamate group is attached to the 3 position of the A ring.
A preferred compound has the formula: 
wherein R denotes a sulphamate group as described above. Here the indicated Me group is vertical.
Preferably, R is the above-mentioned preferred formula for the sulphamate group. In this regard, it is preferred that at least one of R1 and R2 is H.
In formula (II), the oxime group can be of either geometrical isomer form. For example, the oxime group can be the syn isomer. In a preferred embodiment, the oxime group is the anti isomer.
A more preferred compound has the formula: 
wherein R denotes a sulphamate group as described above.
Preferably, R is the above-mentioned preferred formula for the sulphamate group. In this regard, it is preferred that at least one of R1 and R2 is H.
In formula (III), the oxime group can be of either geometrical isomer form. For example, the oxime group can be the syn isomer. In a preferred embodiment, the oxime group is the anti isomer.
Preferably, if the sulphamate group of the compound of the present invention were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activityxe2x80x94i.e. when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37xc2x0 C.
In one preferred embodiment, if the sulphamate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 50 mmoles when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37xc2x0 C.
In another preferred embodiment, if the sulphamate group of the compound were to be replaced with a sulphate group to form a sulphate compound then that sulphate compound would be hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value of less than 50 xcexcmoles when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and 37xc2x0 C.
In a highly preferred embodiment, the compound of the present invention is not hydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2) activity.
The sulphamate compounds of the present invention may be prepared by reacting an appropriate alcohol with the appropriate sulfamoyl chloride, R1R2NSO2Cl.
Preferred conditions for carrying out the reaction are as follows.
Sodium hydride and a sulfamoyl chloride are added to a stirred solution of the alcohol in anhydrous dimethyl formamide at 0xc2x0 C. Subsequently, the reaction is allowed to warm to room temperature whereupon stirring is continued for a further 24 hours. The reaction mixture is poured onto a cold saturated solution of sodium bicarbonate and the resulting aqueous phase is extracted with dichloromethane. The combined organic extracts are dried over anhydrous MgSO4. Filtration followed by solvent evaporation in vacuo and co-evaporated with toluene affords a crude residue which is further purified by flash chromatography.
Preferably, the alcohol is derivatised, as appropriate, prior to reaction with the sulfamoyl chloride. Where necessary, functional groups in the alcohol may be protected in known manner and the protecting group or groups removed at the end of the reaction.
For pharmaceutical administration, the steroid sulphatase inhibitors of this invention can be formulated in any suitable manner utilising conventional pharmaceutical formulating techniques and pharmaceutical carriers, adjuvants, excipients, diluents etc. and usually for parenteral administration. Approximate effective dose rates are in the range 100 to 800 mg/day depending on the individual activities of the compounds in question and for a patient of average (70 Kg) bodyweight. More usual dosage rates for the preferred and more active compounds will be in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day, most preferably from 200 to 250 mg/day. They may be given in single dose regimes, split dose regimes and/or in multiple dose regimes lasting over several days. For oral administration they may be formulated in tablets, capsules, solution or suspension containing from 100 to 500 mg of compound per unit dose. Alternatively and preferably the compounds will be formulated for parenteral administration in a suitable parenterally administrable carrier and providing single daily dosage rates in the range 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250 mg. Such effective daily doses will, however, vary depending on inherent activity of the active ingredient and on the bodyweight of the patient, such variations being within the skill and judgement of the physician.
For particular applications, it is envisaged that the steroid sulphatase inhibitors of this invention may be used in combination therapies, either with another sulphatase inhibitor, or, for example, in combination with an aromatase inhibitor, such as for example, 4hydroxyandrostenedione (4-OHA).
In the method of treatment, the subject is preferably a mammal, more preferably a human. For some applications, preferably the human is a woman.
In summation, the present invention provides novel compounds for use as steroid sulphatase inhibitors, and pharmaceutical compositions containing them. The compounds also have a oestrogenic activityxe2x80x94particularly when compared with EMATE. Thus, the present invention provides novel compounds having steroid sulphatase inhibitory activity which, in some cases, have extremely high activity levels.
In addition, the present invention provides novel compounds having oestrogenic activity which, in some cases, have extremely high activity levels.
It will be appreciated that the present invention also includes the following:
(i) a compound of the present invention or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt;
(ii) one or more processes for the preparation of a compound of the present invention or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt;
(iii) novel intermediates for use in those processes;
(iv) a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt, admixed with a pharmaceutically acceptable diluent, carrier or excipient;
(v) a compound of the present invention, or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof, for use as a medicament;
(vi) the use of a compound of the present invention, or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof, for the manufacture of a medicament for the inhibition of oestrone sulphatase;
(vii) the use of a compound of the present invention, or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof, for the manufacture of a medicament for the inhibition of oestrone sulphatase;
(viii) a method for the inhibition of oestrone sulphatase which method comprises administering to a subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof;
(ix) a method for inhibition of oestrone sulphatase which method comprises administering to a subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt of the compound, or a pharmaceutically acceptable solvate of the compound or the salt or composition thereof.
In the above-mentioned uses and methods, the subject is typically a mammal.
The pharmaceutically acceptable salts of the compounds of/for use in the present invention include suitable acid addition or base salts thereof. For a review on suitable pharmaceutical salts see Berge et al, J Pharm Sci, 66, 1-19 (1977).
By way of example, suitable acid addition salts are formed from acids which form non-toxic salts. Suitable examples of such salts are the hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, benzoate, methanesulphonate, benzenesulphonate and p-toluenesulphonate salts.
Also by way of example, suitable base salts are formed from bases which form non-toxic salts. Suitable examples thereof are the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, N-benzyl-N-(2-phenylethyl)amine, 1-adamantylamine and diethanolamine salts.
As mentioned above, the present invention also covers pharmaceutical compositions comprising the compounds of the present invention. In this regard, and in particular for human therapy, even though the compounds of the present invention (including their pharmaceutically acceptable salts and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.
By way of example, in the pharmaceutical compositions of the present invention, the compounds of the present invention may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Tablets or capsules of the compounds may be administered singly or two or more at a time, as appropriate. It is also possible to administer the compounds in sustained release formulations.
Typically, a physician will determine the dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient.
Alternatively, the compounds of/for use in the present invention can be administered by inhalation or in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. An alternative means of transdermal administration is by use of a skin patch. For example, they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. They can also be incorporated, such as at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
For some applications, preferably the compositions are administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.
The compositions (as well as the compounds alone) can also be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. In this case, the compositions will comprise a suitable carrier or diluent.
For parenteral administration, the compositions are best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
For oral, parenteral, buccal and sublingual administration to subjects (such as patients), the daily dosage level of the compounds of the present invention and their pharmaceutically acceptable salts and solvates may typically be from 10 to 500 mg (in single or divided doses). Thus, and by way of example, tablets or capsules may contain from 5 to 100 mg of active compound for administration singly, or two or more at a time, as appropriate. As indicated above, the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. It is to be noted that whilst the above-mentioned dosages are exemplary of the average case there can, of course, be individual instances where higher or lower dosage ranges are merited and such dose ranges are within the scope of this invention.
Thus the invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, together with a pharmaceutically acceptable diluent, excipient or carrier.
The invention further provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing, for use as a human medicament.
The present invention also provides a veterinary formulation comprising a compound of the present invention, or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, together with a veterinarily acceptable diluent, excipient or carrier.
For veterinary use, a compound of the present invention or a veterinarily acceptable salt thereof, or a veterinarily acceptable solvate of either entity, is typically administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal. However, as with human treatment, it may be possible to administer the compound alone for veterinary treatments.
In addition, the present invention provides a compound of the present invention, or a veterinarily acceptable salt thereof or a veterinarily acceptable solvate of either entity, or a veterinary formulation containing any of the foregoing, for use as an animal medicament. Reference is also made to WO 99/27935, and concurrently-filed U.S. application Ser. No. 09/572,246.
The present invention will now be described only by way of example.
In the examples reference is made to steroid sulphatase inhibition. This is determined according to the teachings of PCT/GB92/01587 wherein the ability of compounds to inhibit oestrone sulphatase activity is assessed using either intact MCF-7 breast cancer cells or placental microsomes. For ease of reference, those teachings are repeated here as Example 1.